Cryptococcus neoformans is a fungal pathogen that infects the human brain. The organism has a defined sexual cycle involving haploid and a strains, and the mating-type is linked to virulence and differentiation. Most environmental and clinical isolates are mating-type, and strains can be more virulent than congenic a strains and preferentially penetrate the CNS during co-infection with a strains by a process involving pheromone signaling similar to quorum sensing in bacteria and other fungi. strains also undergo monokaryotic fruiting and produce basidiospores via a modified sexual cycle. Thus, the structure, function, and evolution of the mating-type locus (MAT) and its links to sexual development, virulence, and ecology are of considerable interest. In studies supported by this award, in aim 1 the MAT locus was cloned, sequenced, and annotated from both mating-types and four divergent Cryptococcus species/varieties, leading to a detailed evolutionary model. In aim 2 on MAT functions, the MAT allele specific genes SXI1 and SXI2a were shown to encode homeodomain proteins that establish cell identity and enable sexual reproduction. MAT encoded genes were shown to play roles in sexual reproduction, virulence, and essential functions. In aim 3 PCR fingerprinting and a MAT locus microarray were developed and provide comparative genomic analysis approaches to elucidate MAT structure and evolution. This progress, combined with recent advances involving genome sequences, microarrays, and discovery of fertile strains and development of congenic strains, allows us to propose new hypotheses and aims. Four interrelated specific aims are proposed. Aim 1 focuses on the sequence of MAT, and extends the analysis to all molecular and mating-types. We propose to sequence MAT from species related to the Cryptococcus pathogenic species cluster; preliminary results indicate this will reveal key evolutionary events that fashioned this large gene complex. Studies to recreate and analyze the hypothesized tripolar intermediate and tetrapolar ancestor by reconfiguring the genomic location of the SXI1/2 genes are also proposed. Aim 2 focuses on the functions of MAT, including roles of pheromones and Ste3 receptor homologs in virulence via processes similar to quorum sensing. Aim 3 focuses on the structure and function of MAT-linked recombinational activators, proposed to have played key roles in MAT origins and evolution. Aim 4 will focus on the structure and evolution of the MAT locus by CGH analysis. These studies will expand understanding of this unique genomic region and its roles in virulence, and serve as a paradigm for gene cluster and sex chromosome evolution and function.